Professional Documents
Culture Documents
KEYWORDS
Rotavirus Cryptosporidium Enterotoxigenic Escherichia coli
Coronavirus Torovirus
Infectious diarrhea remains one the biggest health challenges in both the beef and
dairy industries. More than 20% of beef cattle owners feel that calf diarrhea has a sig-
nificant impact on their economic productivity,1 and diarrhea accounts for more than
half of all calf mortality on dairy farms.2 Currently, enterotoxigenic Escherichia coli
(ETEC), Cryptosporidium parvum, rotavirus, and coronavirus appear to be the most
significant infectious causes of calf diarrhea. Research into the pathophysiology of
these organisms may ultimately lead to more specific treatment and control
recommendations.
Epidemiologic studies of both beef and dairy calves have implicated ETEC as the
major cause of neonatal diarrhea occurring in the first 4 days of life; however it rarely
leads to diarrhea in older calves or adult cattle.3–6 Immediately after birth, oral expo-
sure to fecal coliforms leads to colonization of the gut with the normal commensal
flora, and these organisms continue to move caudally through the gastrointestinal
tract with ingesta.7,8 If environmental contamination is high, ETEC organisms are
ingested at this same time and are able to produce disease caused by the presence
of two virulence factors, K99 fimbria and heat stable toxin. Because nonpathogenic E
coli are extremely common, fecal cultures as a diagnostic test are of little value unless
the presence of these two virulence factors can be demonstrated.
ETEC diarrhea in calves is K99, which is more appropriately referred to as F5.6,11 The
F41 and 987P antigens can also be found in calf ETEC isolates, often in conjunction
with F5.12,13 Because the K99 antigen is only expressed at an environmental pH level
of less than 6.5, the distal small intestine is the initial site of colonization. This is
because the pH level of the intestinal fluid increases as it moves caudally, and it
only reaches this threshold at the ileum.14–18 The ability of K99 ETEC to bind to the
small intestinal epithelium is age dependent and gradually decreases from 12 hours
of age to 2 weeks of age. However, there is not a precipitous drop in the binding ability
that would explain the age resistance to ETEC.19 The attachment of ETEC allows the
bacteria to colonize the ileum, proliferate, and spread proximally through the small
intestine.3,17,18,20 Once established in the gut, ETEC produces heat stable toxin
leading to secretory diarrhea.
Fig. 1. Frame 1: K99 ETEC binds to an intestinal epithelial cell, and heat stable toxin (STa) is
secreted, which binds to the receptor GCC. The enteric nervous system becomes activated by
the secretion of STa, but the mechanism of this activation is unclear. At this point, CFTR is
not active. Frame 2: STa binds to GCC, which converts guanylyl triphosphate (GTP) to
cGMP. cGMP activates cGKII to phosphorylate the CFTR, and the CFTR moves to the luminal
surface and is activated, leading to chloride (Cl) secretion. Frame 3: Secreted STa activates
tyrosine kinase through an unknown pathway, which leads to bicarbonate (HCO3) secretion.
STa also directly inhibits the sodium–hydrogen exchanger, decreasing the movement of so-
dium (Na) and hydrogen (H) across the membrane.
intestine.35,36 The receptor and other messengers in this pathway have not been
elucidated. An opposing model of STa-induced diarrhea has also been proposed
that is not based on fluid secretion caused by movement of chloride or bicarbonate,
but which instead is caused by decreased fluid absorption. In addition to activating
the CFTR, STa inhibits the apical Na-H exchanger, leading to failure of sodium absorp-
tion. This failure of sodium absorption decreases fluid movement from the intestinal
lumen to the interstitial space. The importance of this mechanism of ETEC diarrhea
in calves is unknown because this has been most conclusively shown in the
duodenum and proximal jejunum of rodent models.37,38
Similar to expression of K99, production of STa is pH dependent. When the environ-
mental pH is less than 7.0, toxin production is severely limited.18,39 Therefore, toxin
production is maximized in the distal small intestine because the pH level is greatest
in this segment. Although it has not been directly investigated, it can be theorized that
STa-mediated secretion of bicarbonate and inhibition of the Na-H exchanger would
have the net effect of alkalinizing the proximal small intestine. This would create an
environment more hospitable to ETEC promoting its spread to the proximal small
intestine.
The autonomic and enteric nervous systems are known to be involved in the
secretory response to cholera toxin through the actions of prostaglandin E2 (PGE2),
5-hydroxytriptamine (5-HT), and vasoactive intestinal polypeptide (VIP).40–42 STa-me-
diated secretion may also involve local reflex arcs in the enteric nervous system (ENS);
however, it does not involve the autonomic nervous system.43 Most of the support for
this idea comes from studies that inhibit the ENS and subsequently reduce the
secretory effect of STa.44–46 The neurotransmitters critical in these responses are nitric
oxide (NO) and VIP,40,46 whereas PGE2 and 5-HT are not involved.41 Furthermore,
a well-defined example of the influence of the ENS in ETEC is its role in exacerbating
16 Foster & Smith
CRYPTOSPORDIUM PARVUM
C parvum is one of the most commonly isolated gastrointestinal pathogens from dairy
calves and immunosuppressed humans53 and is a significant cause of waterborne
diarrhea outbreaks.54 Infection occurs when oocysts are ingested from the environ-
ment. Once in the host, the organism goes through a complicated life cycle that
involves multiple stages. The cycle starts with exposure to gastric acid and bile salts,
Calf Diarrhea 17
leading to excystation of the oocyst to the first life stage, the sporozoite. The sporo-
zoites invade the intestinal epithelial cells of the ileum, where the infection is
typically concentrated, but they can infect the gastrointestinal tract anywhere from
the abomasum to the colon. The sprorozoites create an invagination of the luminal
membrane, allowing them to maintain an extracytoplasmic but intracellular location
known as a parasitophorous vacuole. From this location, the sporozoites transform
into trophozoites. At this stage, asexual reproduction occurs and Type I meronts
are formed. Merozoites are then released into the lumen. These organisms can form
additional Type I meronts or Type II meronts, which form micro- and macrogamonts.
Micro- and macrogamonts reproduce sexually to create thin- and thick-walled
oocysts. The thin-walled oocysts lead to autoinfection, whereas the thick-walled oo-
cysts pass out with feces to contaminate the environment. These oocysts are infective
immediately, and remain viable in the environment for extended periods of time.55–57
C parvum oocyst shedding occurs as early as 3 days of age, peaks at 2 weeks of
age, and can continue to occur in adult cattle. However, diarrhea caused by C parvum
rarely occurs after 3 months of age.57–63 After infection, clinical signs peak at 3 to 5
days and last 4 to 17 days.60,64 Some studies have shown that up to 100% of dairy
calves become infected with C parvum,58,65 and become the major source of environ-
mental contamination because calves shed up to 107 oocysts per gram of feces.60
Shedding in beef calves is much less frequent and occurs in less than 5% of calves.59,66
Calves appear to be resistant to subsequent infection after the initial episode of C par-
vum diarrhea.63 Severity of diarrhea and incidence of clinical signs in calves shedding
oocysts can be variable within and between farms, leading some to question the true
importance of C parvum as a primary pathogen;67 however, it has been repeatedly iso-
lated independent of other known pathogens in clinical cases.57
Fig. 2. Normal and C parvum–infected intestinal mucosa from a calf ileum at 100 magnifi-
cation. (A) Normal calf ileal mucosa. (B) and (C). Calf ileal mucosa experimentally infected
with C parvum. Note the blunting of the villi and the hyperplasia of the crypts. There are
more severe histologic changes in (C), because the villi are more atrophied and the mucosa
no longer completely covers the lamina propria (hematoxylin and eosin).
related to the life stage of C parvum, and that apoptosis is inhibited during the tropho-
zoite stage when the organism is most dependent on the host, but then increases later
during the infection. Furthermore, the incidence of apoptosis will vary over time
between infected cells and uninfected neighboring cells. This may be beneficial to
the host to limit spread of the organism, limit the severity of cell loss, and/or speed clear-
ance of the organism.83 Pharmacologically induced apoptosis in infected cell cultures is
also prevented, indicating that apoptosis mechanisms are actively inhibited,78,83 which
has been shown to be mediated by NF-kB.78 Additional research is needed to elucidate
the ultimate beneficiary of this apoptotic regulation: the organism, to maintain its intra-
cellular habitat, or the host, to limit cell loss and spread of infection.
Irrespective of how or why epithelial cell loss and villous atrophy occurs, this leads
to a malabsorptive diarrhea. The net absorption of fluid is caused by the movement of
sodium coupled with either chloride or other nutrients in the villous tip versus the se-
cretion of anions in the crypts. Therefore, absorption is impaired because of the loss of
the mature villous epithelial cells and their associated transporters as well as a de-
crease in total surface area.64,70,84,85 Absorption of sodium and water can still occur
to some degree in the crypts when coupled with glucose or neutral amino acids
(eg, glutamine), which can be used to improve absorption of oral rehydration solu-
tions,86,87 but overall absorption of carbohydrates, lipids, and amino acids is
reduced.88–91 This malabsoption leads to diarrhea that can range from very mild to
life threatening, depending on the dose of organism and coinfection with other
pathogens. However C parvum has not been shown to decrease overall growth in
calves once the infection has resolved.55
Calf Diarrhea 19
Cl /HCO3
C. parvum
+ NaCl
PGE2
Ach/VIP
Ca+/cAMP
PGE2
PGI2
Enteric nerve
Mϕ
Fig. 3. Infection of intestinal epithelial cells with C parvum induces the epithelial cell to se-
crete PGE2 and leads to activation of macrophages (M4) in the lamina propria. This leads to
secretion of PGE2 and PGI2 from the mesenchymal cells. PGI2 activates the enteric nervous
system to secrete acetylcholine (Ach) and VIP. The secretion of Ach, VIP, and PGE2 leads to
an increase in intracellular calcium and cAMP, which activates anion secretion (Cl and
HCO3) and inhibits neutral sodium and chloride absorption (NaCl).
20 Foster & Smith
The mechanism of action of the two prostaglandins differs because PGE2 acts on
the enterocyte directly, whereas PGI2 exerts its effect through the ENS. PGI2 causes
75% of the secretion in C parvum infection by stimulating the nicotinic ganglia and the
VIP-ergic and cholinergic motor neurons that innervate the intestinal mucosa. Prosta-
glandin secretion ultimately leads to increases in calcium and cAMP that increase
anion secretion and decrease sodium absorption.42,70,92 Inhibiting the effects of
prostaglandins on the ENS is a potential method of decreasing the diarrhea associ-
ated with C parvum without exacerbating the villous atrophy. Specifically, peptide
YY, which is naturally found in the intestinal epithelium, is a potent inhibitor of VIP
and can abolish the secretory response to PGI2.97 Furthermore, if the inhibition caused
by prostaglandins can be blocked, the intestine is capable of absorbing NaCl and
water despite the villous atrophy, indicating that the transporters are fully functional,
even in the immature enterocytes.85–87
ROTAVIRUS
Rotavirus was one of the first identified viral causes of diarrhea, and was initially known
as neonatal calf diarrhea virus. Subsequently, it has been found throughout the world
and has been identified as a significant pathogen of children and most other
mammals.114,115 Antibodies to rotavirus can be found in more than 90% of
unvaccinated cattle,116 and the virus was isolated from 94% of dairy calves at a large
dairy and calf ranch during the first 2 weeks of life.117 It has also been isolated from
approximately 20% of calf diarrhea samples,118,119 and from at least one calf on
63% of farms.120 Calves become infected after ingesting the virus from fecal contam-
ination of the environment, because the virus remains quite stable if the temperature
does not get near freezing. The virus typically affects calves less than 3 weeks old, with
a peak incidence at 6 days of age. After ingestion of the virus, the incubation period is
approximately 24 hours, with resolution of diarrhea in uncomplicated cases in 2
days.115 Classically, rotavirus diarrhea is thought to be primarily a malabsorptive
diarrhea, but recent evidence indicates that there is also a toxin-mediated secretory
component as well.
carbohydrates create an osmotic load pulling fluid into the lumen. Furthermore, fluid
secretion from the crypts increases the amount of fluid in the intestinal lumen relative
to villous absorption, which leads to diarrhea.21,114,115 However, the severity of clinical
signs does not always correlate with histologic damage to the villi. This has led to
speculation that there may be another mechanism contributing to the diarrhea seen
with rotaviral infections, and that enterocyte damage is less critical than previously
believed.
In the mid-1990s, a viral enterotoxin was demonstrated to be crucial to the
pathogenesis of rotaviral diarrhea.121 This was the first time an enterotoxin could be
identified in a viral diarrheal pathogen, and this changed our fundamental understand-
ing of rotavirus diarrhea.122 The rotavirus protein, nonstructural glycoprotein 4 (NSP4),
was found to induce a dose- and age-dependent diarrhea that is clinically similar to
rotavirus diarrhea. Unlike the bacterial enterotoxins, diarrhea due to NSP4 is unrelated
to cAMP, cGMP, or CFTR.123,124 The protein is initially produced during intracellular
viral replication and acts on the infected cell. It is secreted or released upon cell death,
and acts in a paracrine manner.125 Exogenous exposure of intestinal epithelial cells to
NSP4 allows binding to caveolae, special lipid rafts within the endoplasmic reticulum
(ER) and cell membrane. It specifically binds to caveolin-1, a transmembrane, hairpin
protein unique to these rafts.126,127 Binding to caveolin-1 leads to an increase in
intracellular calcium concentrations by causing the release of calcium from ER stores
and increasing movement across the plasma membrane. This is mediated by
phospholipase C (PLC), which increases the level of intracellular inositol 1,4,5-triphos-
phate (IP3),124,128–130 however intracellular NSP4 causes the release of calcium
independent of the PLC pathway.131
Extracellular and intracellular exposure to NSP4 causes several changes in the
movement of nutrients and water across the epithelium (Fig. 4). Increases in intracel-
lular calcium inhibit the translocation of disaccharidases from the intracellular vesicles
to the luminal surface, decreasing the ability to digest carbohydrates and leading to
maldigestion and exacerbation of the diarrhea.132–134 NSP4 also directly inhibits
sodium glucose cotransporter SGLT1, the primary sodium and glucose cotransporter
that is critical for effective water absorption, significantly contributing to the pathogen-
esis of rotaviral diarrhea.135 The actions of NSP4 better account for the maldigestion
and malabsorption that are seen in rotavirus diarrhea, and is are likely more important
to the pathogenesis than is histologic damage to the epithelium.
PLC
Glucose IP3
Na+
NSP4
Rotavirus Ca+
Disaccharidases
Enteric nerve
Fig. 4. Once rotavirus replicates in an intestinal epithelial cell, the enterotoxin NSP4 is pro-
duced. It has autocrine effects by causing calcium (Ca) release from the endoplasmic reticu-
lum. NSP4 has paracrine effects by being secreted and binding to caveolin-1. This activates
PLC, which increases cytoplasmic IP3. IP3 increases intracellular calcium by increasing release
from the endoplasmic reticulum and increasing calcium movement across the luminal mem-
brane. The increased intracellular calcium inhibits movement of disaccharidases to the lumi-
nal surface. NSP4 directly inhibits the SGLT1 which decreases the absorption of sodium (Na)
and glucose, and increases chloride (Cl) secretion by an unknown mechanism, but may in-
volve a channel created by NSP4. NSP4 also activates the enteric nervous system by an un-
known mechanism.
CORONAVIRUS
most dry cow vaccinations targeted for neonatal calf diarrhea contain both rotavirus
and coronavirus.
Salmonella is the other major infectious cause of diarrhea in calves, but it is discussed
in a separate article in this issue. A few other minor causes of diarrhea in calves are
worth brief discussion.
Clostridium difficile
Diarrhea caused by Clostridium difficile appears to be an emerging problem in both
humans and veterinary patients. Diarrhea caused by C difficile is mediated by bacterial
toxins that lead to epithelial cell death, damage to epithelial cell tight junctions,
inflammation of the mucosa and submucosa, and activation of the ENS.168 C difficile
and its toxins can be found in the feces of both normal and diarrheic calves,169–172 but
its role as a pathogen has not been clearly established. Purified toxins will cause
epithelial damage and an increase in luminal fluid in a calf intestinal loop model,169
however experimental infection has not been successful.172
Giardia
Giardia organisms can be found in the feces of calves with diarrhea throughout the
world, but is also commonly found in the feces of normal calves.58,59,66,173–182
Some of these studies also found other pathogens along with Giardia, and none
were controlled experiments. Only a single study has documented attempted
experimental infection of calves with Giardia. In that study, histologic changes were
found in only 2 of 12 calves, clinical signs were simply described as not severe, and
26 Foster & Smith
the incidence of diarrhea was not reported.183 Giardia has been documented to cause
villous atrophy in naturally infected calves,184 and is known to cause a malabsorptive
diarrhea in other species.185,186 Therefore, some have proposed that it may not be
a significant cause of disease, but could still negatively impact calf growth.55 This
has also not been experimentally proven. Although Giardia is commonly found in
the feces of both dairy and beef calves, it is unknown if it is truly a pathogen.
Torovirus
In the early 1980s, an infectious agent similar to coronavirus was identified in a herd of
beef cattle in Iowa.187 It was initially named Breda virus, but has subsequently been
renamed torovirus. Since that time, it has been identified in both beef and dairy calves
throughout the world, and 94% of adult cattle are seropositive.188 Torovirus is found in
calves with normal and abnormal feces, but is isolated more frequently in diarrheic
feces. The incidence in calves with diarrhea ranged from 5% to 35%, while it was
never isolated from more than 12% of normal calves. Other pathogens were
commonly, but not always, found in conjunction with torovirus, but none appeared
to be consistently associated with torovirus infection.189–193 After ingestion, the virus
infects the epithelium of the distal half of the jejunum, the ileum, and colon. Histopath-
ologic lesions in experimental infections include necrosis of the crypt and villous
enterocytes and villous atrophy, but infection does not consistently lead to clinical
signs or histologic damage.187,194,195 Although it has not been conclusively shown,
these lesions would be expected to lead to a malabsorptive diarrhea. There is no
specific information on control of torovirus, but as with other viruses, proper housing,
decreasing exposure to adult cattle, and good hygiene are likely important to prevent
its spread.
SUMMARY
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